Glial cell line—derived neurotrophic factor (GDNF) (Lin et al., 1993), neurturin (NTN) (Kotzbauer et al., 1996), persephin (PSP) (Milbrandt et al., 1998) and a recently discovered artemin (ART) (Baloh et al., 1998) form a group of TGF-β family-related neurotrphic proteins. Studies in primary neuronal cultures, as well as in lesioned animal models, have provided evidence that GDNF is a survival factor for embryonic midbrain dopaminergic neurons (Beck et al., 1995; Lin et al., 1993, Tomac et al., 1995), spinal motor neurons (Henderson et al., 1994; Oppenheim et al., 1995; Yan et al., 1995), locus coeruleus noradrenergic neurons (Arenas et al., 1995), and subpopulations of peripheral sensory, sympathetic, and parasympathetic neurons (Buj-Bello et al., 1995; Trupp et al., 1995; reviewed by Airaksinen et al., 1999 and Saarma & Sariola, 1999). The pattern of neurotrophic activity of GDNF is therefore promising for its potential use in the treatment of Parkinson disease, Alzheimer disease, motoneuron diseases and several other neurodegenerative diseases. The biological importance of the GDNF family is illustrated by the phenotype of GDNF null mice which display deficits in primary sensory, sympathetic and motor neurons. These mice also fail to develop kidneys and most of the enteric nervous system and they die at birth (Moore et al., 1996; Pichel et al., 1996; Sanchez et al., 1996).
Despite the potential clinical importance of the GDNF receptor family, the intracellular mechanism of GDNF's action is far from understood. Generally GDNF has been thought to act through a multi-component receptor system including a glycosyl-phosphatidyl-inositol (GPI)-anchored GDNF family receptor α1 (GFRα1) (Jing et al., 1996; Treanor et al., 1996) and a transmembrane receptor tyrosine kinase, Ret (Durbec et al., 1996; Trupp et al., 1996). GFRα1, lacking an intracellular domain, has originally been assessed as a binding site for GDNF, serving only in the presentation of the GFRα1/GDNF complex to Ret (Jing et al., 1996; Treanor et al., 1996; Trupp et al., 1997). There is no doubt that the Ret and GPI-anchored GFRα1 are necessary receptors for GDNF (Cacalano et al., 1998, Enomoto et al., 1998) since mice lacking Ret, GDNF or GFRα1 all share a similar phenotype and die soon after birth. However, it is not known whether these GFRα proteins can evoke intracellular signals upon the action of GDNF family proteins in the absence of Ret.
Ret and GFRα1 expression patterns, although similar, exhibit differences in many tissues (Trupp et al., 1997; Enomoto et al., 1998, Golden et al., 1999, Kokaia et al., 1999), which may be a sign of the distinct signaling from GFRα receptors alone or in conjunction with Ret tyrosine kinase in trans (Yu et al., 1998). We recently showed both in vitro and in vivo (Ylikoski et al., 1998), for example, that GDNF promotes survival of postnatal cochlear sensory neurons expressing GFRα1 mRNA but lacking Ret mRNA. This difference in expression patterns may be a sign of distinct Ret-independent signaling triggered by activation of GFRα receptors.
The triggering of GDNF-dependent intracellular signaling in RN33B cells has also been described (PCT/US96/18197, incorporated herein by reference). RN33B cells were described therein as expressing four putative receptors for GDNF, none of which was c-Ret. Two of the receptors were later determined to be GFRα1 and GFRα2 (reported as GDNFRα and GDNFRβ, respectively, U.S. patent application Ser. No. 08/861,990, incorporated herein by reference). The mechanism of the Ret-independent signaling, however, was not known or described.
Although GPI-anchored membrane proteins have not been conclusively shown to exhibit independent intracellular signaling functions, evidence suggesting this possibility has been increasing (Simons and Ikonen, 1997; Friedrichson and Kurzchalia, 1998; Harder et al., 1998; Varma and Mayor, 1998; Viola et al., 1999). It has been shown, for example, that GPI-anchored: proteins in the immune system can mediate intracellular signaling events, such as activation of the small G-proteins, Src-type tyrosine kinases and elevation of intracellular free calcium concentration ([Ca2+]i) (Green et al., 1997; Brown and London, 1998; Viola et al., 1999). GPI-anchored independent signaling has not previously been shown in cells of the nervous system, however.
The aim of the invention, therefore, is to further elucidate Ret independent intracellular signaling. We specifically address the role of GDNF-activated signaling in dorsal root ganglion (DRG) neurons isolated from Ret-null (Ret−/−) transgenic mice (Schuchardt et al., 1994) and in other Ret-negative cell lines, for the purpose of developing a method for identifying compounds which are agonists or antagonists of Ret independent signaling.